Abstract 6060: A symmetrical hexavalent trispecific antibody, HH160, targeting PD-1, CTLA-4 and VEGF-A for enhanced anti-cancer effects

Abstract Background: Antiangiogenic agents, such as the anti-vascular endothelial growth factor A (VEGF-A) antibody Bevacizumab, are among the first targeted therapies in cancer treatment. Immune checkpoint inhibitors (ICIs), including anti-PD-1/L1 and anti-CTLA-4 antibodies, have revolutionized cancer therapy. Combinations of anti-VEGF agents and ICIs have demonstrated enhanced efficacy in treating various types of cancers, including liver cancer, non-small cell lung cancer, and cervical cancer, indicating synergistic anti-tumor mechanisms. Here, we report a novel symmetrical hexavalent trispecific antibody, HH160, which targets PD-1, CTLA-4, and VEGF-A, aiming to enhance therapeutic effects, increase dosing convenience, achieve tumor-specific drug distribution, and reduce treatment-related side effects. Methods: HH160 was constructed by fusing anti-PD-1 and anti-CTLA-4 nanobodies to the N- and C-termini of the heavy chain of an anti-VEGF-A human IgG1 antibody with a silent Fc, forming a symmetrical hexavalent trispecific antibody. Binding affinity was assessed using Surface Plasmon Resonance, and the EC50 and IC50 were determined using ELISA and FACS. Immune-related cellular functions were evaluated using luciferase-based reporter assays and Mixed Lymphocyte Reaction (MLR) assays. CTLA-4 mediated cellular internalization was evaluated in Jurkat-PD-1/CTLA-4 cells, and pharmacokinetics (PK) in mice were also assessed. The anti-tumor efficacy of HH160, including anti-tumor immune functions and anti-angiogenesis were assessed respectively in syngeneic hPD-1/hCTLA-4 humanized C57BL/6 mouse tumor models and immunodeficient mice bearing VEGF-A-expressing cells. Results: HH160 exhibited strong binding to the three targets—PD-1, CTLA-4 and VEGF-A— and effectively blocked their ligand interactions. HH160 showed strong stimulation of IL-2 and IFN-γ secretion in MLR assays, indicating enhanced anti-tumor immune activity and the potential to overcome resistance to single-targeting agents. HH160 was efficiently internalized in Jurkat-PD-1/CTLA-4 cells via receptor-mediated internalization, suggesting its potential to reduce the VEGF-A levels in the tumor microenvironment. HH160 also exhibited a favorable PK profile in BALB/c mice. Significant immune-mediated and anti-angiogenesis-mediated anti-tumor effects were observed in hPD-1/hCTLA-4 humanized mouse tumor models and immunodeficient mice bearing VEGF-A-expressing cells. Conclusions: Our study demonstrates that HH160 is a promising trispecific antibody for simultaneously targeting PD-1, CTLA4 and VEGF-A. It has the potential to harness the synergistic anti-tumor mechanisms, overcome resistance to monotherapy, and simplify the dosing regimen, supporting further development and clinical investigation. Citation Format: Chunmei Liu, Jianhe Chen, Fang Yang, Fangfang Ren, Jing Shao, Juan Liu, Bin Ye, Jianhua Sui. A symmetrical hexavalent trispecific antibody, HH160, targeting PD-1, CTLA-4 and VEGF-A for enhanced anti-cancer effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6060.